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Type III secretion- the various functions of the translocon operon in bacterial pathogenesis
Umeå University, Faculty of Medicine, Molecular Biology.
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In order to establish colonisation of a human host, pathogenic Yersinia use a type III protein secretion system to directly intoxicate host immune cells. Activation of this system requires target cell contact and is a highly regulated process. Both the intoxication and regulation events depend on the lcrGVHyopBD translocon operon, which is highly conserved in many bacterial pathogens. In this study, the role of individual operon members was analysed and functional domains identified by using the highly homologous pcrGVHpopBD operon of P. aeruginosa as a comparative tool.

Yersinia spp. and P. aeruginosa were shown to form translocation pores of a similar size that promoted equally efficient protein delivery. A strong dependency on interactions between native translocator(s) in protein delivery was revealed, suggesting that each pathogen has delicately fine-tuned this process to suit its own infection niche. In particular, the C-terminus of YopD was shown to possess functional specificity for effector delivery in Yersinia that could not be conferred by the comparable region in homologous PopD. Moreover, a role for LcrV and PcrV in substrate recognition during the protein delivery process was excluded.

The N-terminus of LcrH was recognized as a unique regulatory domain, mediating formation of LcrH-YscY regulatory complexes in Yersinia, while equivalent complexes with analogous proteins were not formed in P. aeruginosa. These results compliment the idea that a negative regulatory pathway involving LcrH, YopD, LcrQ and YscY is unique to Yersinia.

Finally, PcrH was identified as a new member of the translocator class of chaperones, being essential for assembly of a functional PopB/PopD mediated translocon in P. aeruginosa. However, in contrast to the other members of this family, PcrH was dispensable for type III regulation. Moreover, both LcrH and PcrH were shown to possess tetratricopeptide repeats crucial for their chaperone function. One tetratricopeptide repeat mutant in LcrH was even isolated that failed to secrete both YopB and YopD substrates, even though stability was maintained. This demonstrates for the first time that LcrH has a role in substrate secretion in addition to its critical role in promoting substrate stability.

Place, publisher, year, edition, pages
Umeå: Molekylärbiologi , 2004. , 82 p.
Keyword [en]
Molecular biology, Yersinia, Pseudomonas aeruginosa, type III secretion, chaperone, translocation, regulation, lcrGVHyopBD, pcrGVHpopBD
Keyword [sv]
Molekylärbiologi
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-331ISBN: 91-7305-712-6 (print)OAI: oai:DiVA.org:umu-331DiVA: diva2:143130
Public defence
2004-11-05, Major Groove, 6L, NUS, Umeå Universitet, 90187, Umeå, 09:00
Opponent
Supervisors
Available from: 2004-10-06 Created: 2004-10-06Bibliographically approved
List of papers
1. Comparative analysis of type III effector translocation by Yersinia pseudotuberculosis expressing native LcrV or PcrV from Pseudomonas aeruginosa.
Open this publication in new window or tab >>Comparative analysis of type III effector translocation by Yersinia pseudotuberculosis expressing native LcrV or PcrV from Pseudomonas aeruginosa.
2003 (English)In: Journal of Infectious Diseases, ISSN 0022-1899, E-ISSN 1537-6613, Vol. 188, no 2, 239-249 p.Article in journal (Refereed) Published
Abstract [en]

The homologues LcrV of Yersinia species and PcrV of Pseudomonas aeruginosa are pore-forming components. When expressed in a Yersinia lcrV background, PcrV formed smaller pores in infected erythrocyte membranes, correlating to a lowered translocation of Yersinia effectors. To understand this phenomenon, cytotoxins exoenzyme S of P. aeruginosa and YopE of Yersinia were introduced into a Yersinia background without Yop effectors but expressing LcrV or PcrV. Comparable translocation of each substrate indicated that substrate recognition by LcrV/PcrV is not a regulator of translocation. Yersinia harboring pcrV coexpressed with its native operon efficiently translocated effectors into HeLa cell monolayers and formed large LcrV-like pores in erythrocyte membranes. Thus, a PcrV complex with native P. aeruginosa translocon components is required to form fully functional pores for complete complementation of effector translocation in Yersinia.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-4144 (URN)10.1086/376452 (DOI)12854079 (PubMedID)
Available from: 2004-10-06 Created: 2004-10-06 Last updated: 2017-12-14Bibliographically approved
2. Dissection of homologous translocon operons reveals a distinct role for YopD in type III secretion by Yersinia pseudotuberculosis.
Open this publication in new window or tab >>Dissection of homologous translocon operons reveals a distinct role for YopD in type III secretion by Yersinia pseudotuberculosis.
2003 (English)In: Microbiology, ISSN 1350-0872, E-ISSN 1465-2080, Microbiology, Vol. 149, no 9, 2615-2626 p.Article in journal (Refereed) Published
Abstract [en]

The homologous pcrGVHpopBD and lcrGVHyopBD translocase operons of Pseudomonas aeruginosa and pathogenic Yersinia spp., respectively, are responsible for the translocation of anti-host effectors into the cytosol of infected eukaryotic cells. In Yersinia, this operon is also required for yop-regulatory control. To probe for key molecular interactions during the infection process, the functional interchangeability of popB/yopB and popD/yopD was investigated. Secretion of PopB produced in trans in a yopB null mutant of Yersinia was only observed when co-produced with its native chaperone PcrH, but this was sufficient to complement the yopB translocation defect. The Yersinia yopD null mutant synthesized and secreted PopD even in the absence of native PcrH, yet this did not restore YopD-dependent yop-regulatory control or effector translocation. Thus, this suggests that key residues in YopD, which are not conserved in PopD, are essential for functional Yersinia type III secretion.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-4145 (URN)10.1099/mic.0.26322-0 (DOI)12949185 (PubMedID)
Available from: 2004-10-06 Created: 2004-10-06 Last updated: 2017-12-14Bibliographically approved
3. PcrH of Pseudomonas aeruginosa is essential for secretion and assembly of the type III translocon
Open this publication in new window or tab >>PcrH of Pseudomonas aeruginosa is essential for secretion and assembly of the type III translocon
2003 (English)In: Journal of Infectious Diseases, ISSN 0022-1899, E-ISSN 1537-6613, Vol. 188, no 12, 1909-1921 p.Article in journal (Refereed) Published
Abstract [en]

Pseudomonas aeruginosa harbors a type III secretion system that translocates antihost effectors into an infected eukaryotic cell. PcrH is a key component of type III secretion in this essential virulence strategy. In the absence of PcrH, P. aeruginosa is translocation deficient because of a specific reduction in presecretory stability and subsequent secretion of PopB and PopD, 2 proteins essential for the translocation process. PcrH exerts this chaperone function by binding directly to PopB and PopD. Consistent with the genetic relatedness of PcrH with LcrH of pathogenic Yersinia species, these proteins are functionally interchangeable with respect to their ability to complement the translocation defect associated with either a lcrH or pcrH null mutant, respectively. Thus, the translocator class of chaperones performs a critical function in ensuring the assembly of a translocation competent type III secreton. Finally, unlike the regulatory roles of other translocator-class chaperones (e.g., LcrH, SicA of Salmonella enterica, and IpgC of Shigella species), in vitro regulation of P. aeruginosa type III secretion does not involve PcrH.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-4146 (URN)10.1086/379898 (DOI)14673772 (PubMedID)
Available from: 2004-10-06 Created: 2004-10-06 Last updated: 2017-12-14Bibliographically approved
4. Characterization of the tetratricopeptide repeats in type III secretion chaperones- mediators of substrate binding and specificity.
Open this publication in new window or tab >>Characterization of the tetratricopeptide repeats in type III secretion chaperones- mediators of substrate binding and specificity.
Show others...
Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-4147 (URN)
Available from: 2004-10-06 Created: 2004-10-06 Last updated: 2010-01-13Bibliographically approved
5. Mapping of an YscY binding regulatory domain within the type III secretion chaperone LcrH of Yersinia pseudotuberculosis.
Open this publication in new window or tab >>Mapping of an YscY binding regulatory domain within the type III secretion chaperone LcrH of Yersinia pseudotuberculosis.
Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-4148 (URN)
Available from: 2004-10-06 Created: 2004-10-06 Last updated: 2010-01-13Bibliographically approved

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